Table 1 PCR primers and thermal cycling conditions

BSP

Forward: 5'-AAGAAAAGAAAAGAAAAGAAAATTTAG-3'

Reverse: 5'-TTTAGTGAGGTAGATAATTTTTT-3'

PCR conditions

Initial denaturation at 95°C (10 min) and 35 cycles at 95°C (3 s), 52°C (3 s), 72°C (5 s), and a final extension at 72°C (10 s)

MSP

Methylation-

Forward: 5'-TAGATAATTTTTTTTAGATTTTGCGGTCG-3'

Specific

Reverse: 5'-TCCTCGCGAAACTACGC-3'

PCR condition

Initial denaturation at 95°C (10 min) and annealing temperature decrement of 0.5°C every cycle (from 70°C to 66.5°C) followed by 32 cycles of 66°C (3 s), 72°C (5 s), and a final extension at 72°C (10 s)

MSP

Non-methylation

Forward: 5'-TAGATAATTTTTTTTAGATTTTGTGGTTG-3'

-specific

Reverse: 5'-TCCTCACAAAACTACAC-3'

PCR condition

Initial denaturation at 95°C (10 min) and annealing temperature decrement of 0.5°C every cycle (from 64°C to 60.5°C) followed by 32 cycles of 60°C(3 s), 72°C (5 s), and a final extension at 72°C (10 s)